Saturated fatty acid– and/or monounsaturated fatty acid–containing phosphatidic acids selectively interact with heat shock protein 27

Diacylglycerol kinase (DGK) α, which is a key enzyme in the progression of cancer and, in contrast, in T-cell activity attenuation, preferentially produces saturated fatty acid (SFA)– and/or monounsaturated fatty acid (MUFA)–containing phosphatidic acids (PAs), such as 16:0/16:0-, 16:0/18:0-, and 16:1/16:1-PA, in melanoma cells. In the present study, we searched for the target proteins of 16:0/16:0-PA in melanoma cells and identified heat shock protein (HSP) 27, which acts as a molecular chaperone and contributes to cancer progression. HSP27 more strongly interacted with PA than other phospholipids, including phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, cardiolipin, phosphatidylinositol, phosphatidylinositol 4-monophosphate, and phosphatidylinositol 4,5-bisphosphate. Moreover, HSP27 is more preferentially bound to SFA- and/or MUFA-containing PAs, including 16:0/16:0- and 16:0/18:1-PAs, than PUFA-containing PAs, including 18:0/20:4- and 18:0/22:6-PA. Furthermore, HSP27 and constitutively active DGKα expressed in COS-7 cells colocalized in a DGK activity–dependent manner. Notably, 16:0/16:0-PA, but not phosphatidylcholine or 16:0/16:0-phosphatidylserine, induced oligomer dissociation of HSP27, which enhances its chaperone activity. Intriguingly, HSP27 protein was barely detectable in Jurkat T cells, while the protein band was intensely detected in AKI melanoma cells. Taken together, these results strongly suggest that SFA- and/or MUFA-containing PAs produced by DGKα selectively target HSP27 and regulate its cancer-progressive function in melanoma cells but not in T cells.

In the present study, to explore how DGKα plays reverse roles in cancer cells and T lymphocytes, we searched for the target proteins of 16:0/16:0-PA in human melanoma cells. We identified heat shock protein (HSP) 27, which acts as a molecular chaperone and is a biomarker of cancer (36), as a novel SFA-and/or MUFA-containing PA (SFA/MUFA-PA)-binding protein. Moreover, 16:0/16:0-PA induced oligomer dissociation of HSP27, which is an indication of its activation. Furthermore, constitutively active DGKα recruited HSP27 to the plasma membrane and colocalized it in a DGK activity (PA)-dependent manner. Intriguingly, HSP27 protein was barely expressed in Jurkat T cells, while the protein was enriched in AKI melanoma cells. Therefore, these results strongly suggest that SFA-and/or MUFA-containing PA species generated by DGKα interact with HSP27 and selectively regulate its cancer-progressive function in melanoma cells but not in T cells.
HSP27 is a molecular chaperone and is known to be a biomarker of cancer, renal injury and fibrosis, and neurodegenerative and cardiovascular disease (36). In particular, the levels of HSP27 are increased in hepatocellular carcinoma cells, and moreover, HSP27 promotes proliferation and invasion, which consequently confer aggressiveness to cancer cells (37,38).
Next, we cloned human HSP27 cDNA (accession number: AB020027) from the mRNAs of AKI cells and ligated it with the pET-28a vector. 6× His-tagged HSP27 protein was produced in Escherichia coli cells and purified by affinity chromatography using nickel-nitrilotriacetic acid agarose. We confirmed that 6× His-HSP27 was detected as a single band with a molecular mass of approximately 30 kDa, which was recognized by an anti-6× His antibody and was thus successfully purified (Fig. 1C).
The PA-binding activity of clathrin coat assembly protein AP180 was affected by liposome diameters (39). Therefore, the lipid binding activities of HSP27 were determined using liposomes with different diameters (100 nm and 1000 nm). HSP27 intensely bound to both sizes (100 nm (Fig. 4, A and B) and 1000 nm (Fig. 4, C and D)) of liposomes containing 16:0/16:0-PA), and the binding activity was stronger than those of 16:0/ 16:0-PS-and PC-containing liposomes (Fig. 4, A-D). No substantial differences between 100 nm and 1000 nm liposomes were observed (Fig. 4, A-D). Therefore, it is likely that different membrane curvatures and shapes formed by various liposome diameters fail to substantially affect the interaction of HSP27 with PA.

HSP27 most strongly binds to PA among various lipids
To measure the lipid binding selectivity of HSP27 in more detail, we carried out a lipid overlay assay using a nitrocellulose membrane spotted with diverse lipids that included 16:0 as fatty acid chains. HSP27 exhibited an intense interaction with 16:0/16:0-PA (Fig. 5A)  , and cardiolipin (CL), which are acidic lipids, are also associated with HSP27 (Fig. 5A). However, their binding intensities were lower than that of PA (Fig. 5B). Therefore, these results indicate that HSP27 selectively and most intensely binds to PA.

HSP27 colocalizes with constitutively active DGKα in cells
We next investigated whether HSP27 can associate with PA in cells. When EGFP-DGKα-CA (a constitutively active mutant lacking EF-hand motifs (Δ1-196)), which produces ultracentrifugation. SDS-PAGE (15% acrylamide) was conducted, and separated proteins were detected by Western blotting using an anti-HSP27 antibody. The position of endogenous HSP27 is indicated with a black arrowhead. B, D, and H, The amounts of protein in the supernatant (s) and precipitate (p) were quantified by densitometry using ImageJ software. Binding activity was calculated as the percentage of the precipitate band intensity compared to the total band intensity. Values are presented as the mean ± SD of three independent experiments. *p < 0.05, ***p < 0.005, one-way ANOVA followed by Tukey's post hoc test. ANOVA, analysis of variance; HSP, heat shock protein; PA, phosphatidic acid; PC, phosphatidylcholine; PS, phosphatidylserine; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.  (29), was expressed in COS-7 cells, the constitutively active mutant was located at the plasma membrane (Fig. 8, A-C). Moreover, mCherry-HSP27 coexpressed with EGFP-DGKα-CA showed a higher plasma membrane/ cytosol ratio of HSP27 at the membrane region where DGKα-CA is colocalized with HSP27 (3.1) than mCherry-HSP27 coexpressed with EGFP alone (1.6) (Fig. 8, A-C). Notably, the plasma membrane/cytosol ratio (2.4) of HSP27 was significantly attenuated by CU-3 (10 μM), a DGKα-selective inhibitor (29), (Fig. 8, A and C), although DGKα-CA was located at the plasma membrane even in the presence of CU-3 (Fig. 8, A and B). These results indicate that the colocalization of EGFP-DGKα-CA with HSP27 was markedly reduced by CU-3. In addition, although EGFP-DGKα-CA-KD, a kinasedead inactive mutant of DGKα-Δ1-196 in which Gly-435 is substituted with Asp (43), was located at the plasma membrane (plasma membrane/cytosol ratio: 4.0) (Fig. 8, D and E), plasma membrane/cytosol ratio (3.0) of mCherry-HSP27 coexpressed with the inactive mutant was lower than that Binding activity was calculated as the percentage of the precipitate band intensity compared to the total band intensity (100 nm (B) and 1000 nm (D)). Values are presented as the mean ± SD of three independent experiments. ***p < 0.005, one-way ANOVA followed by Tukey's post hoc test. ANOVA, analysis of variance; HSP, heat shock protein; PA, phosphatidic acid; PC, phosphatidylcholine; PS, phosphatidylserine; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.  Figure 5. Binding activity of 6×His-HSP27 to various lipids. A, Lipid overlay assay of 6×His-HSP27 using various lipids. Equimolar amounts (100 pmol) of various lipids were spotted onto nitrocellulose membranes (Lipid Strips, Echelon Biosciences) as indicated. The acyl chain(s) of these glycerolipids and sphingolipid are C16:0. The membrane was incubated with purified 6×His-HSP27 (20 nM). Lipid-bound proteins were detected with an anti-6×His antibody. The data shown are representative of three independent experiments that gave similar results. B, Spot intensities were quantified by densitometry using ImageJ software. The binding activity (spot intensity) of HSP27 to PA was set to 100%. Values are presented as the mean ± SD of three independent experiments. *p < 0.05, ***p < 0.005 versus PA, one-way ANOVA followed by Tukey's post hoc test. ANOVA, analysis of variance; Chol, cholesterol; CL, cardiolipin; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PI(4)P, phosphatidylinositol-4-monophosphate; PI(4,5)P 2 , phosphatidylinositol-4,5bisphosphate; PI(3,4,5)P 3 , phosphatidylinositol-3,4,5-trisphosphate; SGC, 3sulfogalactosylceramide; SM, sphingomyelin; TG, triglyceride.
(4.0) with EGFP-DGKα-CA (Fig. 8, D and F). These results indicate that the inactive mutant was less strongly colocalized with mCherry-HSP27 than EGFP-DGKα-CA. Therefore, these results indicate that the colocalization between HSP27 and DGKα occurs in a DGK activity (PA production)-dependent manner, suggesting that HSP27 can interact with PA produced by DGKα in cells.

HSP27 is highly expressed in AKI melanoma cells but not Jurkat T cells
To analyze whether HSP27 is substantially expressed in T cells, where DGKα plays roles different from cancer cells (6,34), we next examined the expression levels of HSP27 in Jurkat T cells by Western blotting. We found that the HSP27 protein was barely detectable in Jurkat T cells (less than 10% of AKI melanoma cells) (Fig. 10, A and B), although the protein band was strongly detected in AKI melanoma cells (Fig. 10, A  and B), suggesting that SFA/MUFA-PAs function via HSP27 only in melanoma cells but not in T cells.

Discussion
In the present study, we demonstrated for the first time that a chaperone, HSP27, is a novel PABP that prefers SFA-and/or MUFA-containing PA species. Several PABPs have been reported to recognize different PA species (11)(12)(13)(14)(15). HSP27 was newly added to the list, which is still growing. Active DGKα recruited and colocalized with HSP27 at the plasma membrane in a DGK activity (PA)-dependent manner. Notably, 16:0/16:0-PA induced oligomer dissociation of HSP27, which enhances its chaperone activity.
Essentially the same results (PA, PC, and PS binding) were obtained by the liposome sedimentation (Figs. 2 and 4) and lipid overlay (Fig. 5) assays. However, high background activities, probably due to hydrophobic interaction, were observed in the liposome sedimentation assay. It is likely that, in the lipid overlay assay, HSP27 mainly recognizes the hydrophilic polar head of PA. However, in the liposome sedimentation assay, HSP27 probably distinguishes fatty acid moieties (hydrophobic region) of PA in liposomes and/or different circumstances on liposome surfaces including different density of lipids generated by distinct fatty acid composition of PAs in addition to the polar head of PA.
The amounts of PS are higher than those of PA in cellular membranes. PS liposomes cosedimented HSP27 (Fig. 2, G and  H). However, the sedimentation activity is almost the same as ultracentrifugation. SDS-PAGE (15% acrylamide) was performed, and separated proteins were detected by Western blotting with anti-6×His antibody (C) or stained with Coomassie Brilliant Blue (E and G). D, F, and H, the amounts of protein in the supernatant (s) and precipitate (p) were quantified by densitometry using ImageJ software. Binding activity was calculated as the percentage of the precipitate band intensity compared to the total band intensity. Values are presented as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.005, one-way ANOVA followed by Tukey's post hoc test. ANOVA, analysis of variance; HSP, heat shock protein; PA, phosphatidic acid; PC, phosphatidylcholine; PS, phosphatidylserine; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.  PC-binding activity (Fig. 2, G and H). Moreover, the PS-binding activity was not detected in the lipid overlay assay (Fig. 5). Indeed, HSP27 is not localized to the plasma membrane where PS is enriched (Fig. 8). Moreover, PS did not induce oligomer dissociation of HSP27 (Fig. 9). These results allow us to speculate that PS-binding activity of HSP27 is substantially lower than its PA-binding activity and does not activate HSP27 in cells.
HSP27 binds to SFA/MUFA-PA more strongly than to PUFA-PA (Fig. 6). There is only one report indicating that a PABP, creatine kinase muscle type, prefers SFA/MUFA-PAs (51). In contrast to these PABPs, PDE4A1, Opi1p, and sporulation-specific protein 20p failed to exhibit substantial preference among PA molecular species (49). It is possible that, in addition to DGKα, other PA-producing enzymes such as lysoPA acyltransferase and phospholipase D (PLD) also affect HSP27 function. Indeed, lysoPA acyltransferase isoform expression has been shown to enhance the proliferation of cancer cells and correlates with an increased risk of tumor development and aggressiveness of tumors (58). Moreover, increased expression of PLD enzymes (PLD1 and PLD2) has been implicated as contributing factors in several types of human cancer, and the role of PLD in pathways involved in cancer progression and tumorigenesis has been reported (59).
PA binding may have effects similar to Ser/Thr phosphorylation because both PA binding and phosphorylation introduce negative charge(s) of a phosphate group to the protein. It was reported that HSP27 is phosphorylated by mitogenactivated protein kinase (MAPK)-activated protein kinase (MAPKAPK) 2 and 3, MAPKAPK5, PKC, cGMP-dependent kinase, Akt/protein kinase B, and protein kinase D (61). The activities of PKCδ (62) and ε (63, 64) are enhanced by PA in addition to DG. Akt/protein kinase B (65, 66) and C-Raf upstream of MAPK (extracellular signal-regulated kinase (ERK)) (67-69) are also activated by PA. Therefore, it is possible that PA synergistically induces the dissociation of HSP27 oligomers via direct binding to HSP27 and activation of HSP27 phosphorylation pathways.
Overexpressed DGKα recruited HSP27 to the plasma membrane in COS7 cells in a DGK activity (PA)-dependent manner (Fig. 8), suggesting that SFA-and/or MUFAcontaining PA species generated by DGKα bind to and recruit HSP27 to the plasma membrane and activate the protein. DGKα is highly expressed in cancer cells, such as melanoma (23) and hepatocellular carcinoma (24) cells, but not in normal melanocytes or hepatocytes. Therefore, it is possible that SFA-and/or MUFA-containing PAs produced by abundant DGKα can interact with and activate HSP27 in cancer cells as well and, consequently, induce cancer cell proliferation and cancer progression. DGKα commonly generates SFA-and/or MUFA-containing PA species, for example, 16:0/16:0-PA in melanoma and T cells (30,35). However, HSP27 is barely expressed in Jurkat T cells (Fig. 10), while HSP27 is also enriched in cancer cells (37,38). Moreover, the human protein atlas showed that HSP27 is not expressed in lymph node and spleen (https://www.proteinatlas.org/ ENSG00000106211-HSPB1/tissue). Therefore, this expression pattern may explain at least in part how DGKα plays reverse roles in T cells (attenuator) and cancer cells (enhancer) (6,34). However, further studies are required to elucidate signal transduction through the DGKα-16:0/16:0-PA-HSP27 pathway during cancer cell proliferation and cancer progression more in detail and how DGKα has cancer-and T-cellselective functions.
Elevated levels of DGKα and PA are related to cancer initiation and progression (23,34,71,72). DGKα prevents apoptosis through the PKCζ-NF-κB pathway in melanoma cells (23). Moreover, DGKα promotes hepatocellular carcinoma proliferation via activation of the Ras-Raf-MAPK/ERK kinase-ERK pathway (24). Furthermore, this isozyme inhibits apoptosis of glioblastoma and melanoma cells through the PDE4A1-cAMP-mTOR pathway (32). PKCζ (73), C-Raf (67)(68)(69), and PDE4A1 (49,74) are activated by PA. Therefore, it is likely that PA regulates the activities of these enzymes in the signaling pathways. In addition to these enzymes, our results showed that PA targets HSP27, which prevents apoptosis and promotes proliferation in cancer cells. Therefore, it is possible that DGKα utilizes multiple pathways to promote the aggressiveness of cancer cells.
In summary, in the present study, we demonstrated that SFA/MUFA-PAs, which are produced by DGKα in cancer cells (30), strongly bind to HSP27, which is highly expressed in melanoma cells but not in T cells and attenuates its oligomer formation. Our results shed light on a novel function of SFA/ SFA-and/or MUFA-containing PAs bind to and dissociate HSP27 MUFA-PA and allow us to speculate about the functional linkage between the pro-cancer proteins, HSP27 (37,38) and DGKα (33,34).   (20 mol%)]. For the lipid-binding assay, the combined dried lipid mixture was hydrated at 95 C in HEPES buffer for 45 min and vortexed for 1 min once every 15 min during hydration. The liposomes were then subjected to five freezethaw cycles (−196 C for 3 min, 95 C for 3 min). Liposomes were formed by sonication at 90 C using a Branson Sonifier 450, or the liposomes were further extruded 11 times through a 100 nm or 1000 nm polycarbonate membrane using a Mini Extruder (Avanti Polar Lipids) (77). The extruder was brought to 95 C prior to extrusion. Since the lipid forms a bilayer, half of the actual concentration was considered (78).

Experimental procedures
Identification of HSP27 as a PA-binding protein AKI cells were washed two times with phosphate-buffered saline and lysed in HEPES buffer containing 25 mM HEPES (pH 7.4), 100 mM NaCl, and 1 mM dithiothreitol. After sonication, insoluble materials were removed by ultracentrifugation (200,000g for 30 min at 4 C). AKI cell lysates were incubated with the PC liposomes at 4 C for 30 min, and nonspecific protein bound to the vesicles were removed by centrifugation at 200,000g for 1 h at 4 C. The resultant supernatant was incubated with the 16:0/16:0-PS or 16:0/16:0-PA liposomes at 4 C for 30 min and then centrifuged at 200,000g at 4 C for 1 h. The precipitates were dissolved in HEPES buffer containing 25 mM HEPES, pH 7.4, 100 mM NaCl, and 1 mM dithiothreitol. The 16:0/16:0-PAbinding proteins were separated by SDS-PAGE and visualized by silver staining (Fig. 1). In-gel digestion and LC-MS/MS identification of proteins in the 28 kDa bands were carried out as previously described (79). Desalted tryptic peptides were analyzed by an Ultimate 3000 RSLCnano system (Thermo Fisher Scientific, Waltham, MA, USA) coupled to a Q Exactive hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific) equipped with a nano ESI source. The protein identification was performed using PEAKS XPro (PEAKS Studio 10.6 build 20201015; Bioinformatics Solutions Inc. Waterloo, Ontario, CA). The analytical parameters were set as follows: search engine, sequest HT; protein database, Swissprot (Homo Sapiens); enzyme name, trypsin; parent mass tolerance, 10.0 ppm; false discovery rate <0.01; unique peptides ≥ 2. Proteins belonging to keratin were excluded from the identification results as contaminants. Based on triplicate experiments, we targeted the protein of average mass, 28 kDa, which was reproducibly identified in the PA-liposomebinding fraction.

Reverse transcription PCR, protein expression, and purification of HSP27
AKI cells were washed twice with phosphate-buffered saline (pH 7.4) and collected by centrifugation (500g, 4 C, 3 min), and total RNA was isolated as previously described (80). cDNA was generated using Transcriptor reverse transcriptase (Roche Diagnostics, Mannheim, Germany).

Lipid overlay assay
One hundred picomoles of various lipids was spotted in a nitrocellulose membrane (Lipid Strips; Echelon Biosciences). The membranes were blocked with 2% skim milk in phosphate-buffered saline (pH 7.4) for 1 h at 4 C. After blocking, 10 ml of 3% fatty acid-free bovine serum albumin and 0.1% Tween 20 in phosphate-buffered saline (pH 7.4) containing 6× His-tagged HSP27 (final concentration: 20 nM) was added to the membranes. The membrane was incubated for 20 min at 4 C and was then incubated with an anti-6× His antibody for 1 h at 4 C, followed by incubation with antimouse IgG conjugated with horseradish peroxidase (Bethyl Laboratories, Montgomery, TX, USA) antibody. Finally, lipidbound 6× His-HSP27 was visualized using an enhanced chemiluminescence Western blotting detection system (GE Healthcare, Little Chalfont, UK).

Liposome-binding assay
The purified 6× His-tagged HSP27 protein (final concentration: 0.5 μM) was dissolved in HEPES buffer and incubated with the PA-containing or control liposomes at 4 C for 30 min. Samples were ultracentrifuged at 200,000g at 4 C for 1 h. The precipitate was dissolved in HEPES buffer containing 25 mM HEPES (pH 7.4), 100 mM NaCl, and 1 mM dithiothreitol. SDS-PAGE (15% acrylamide) was conducted, and separated proteins were stained with Coomassie Brilliant Blue or detected by Western blotting using an anti-6× His antibody.
To measure the binding activity between PA-containing liposomes and endogenous HSP27, AKI cells were washed two times with phosphate-buffered saline and lysed in HEPES buffer containing 25 mM HEPES (pH 7.4), 100 mM NaCl, and 1 mM dithiothreitol by sonication. After sonication, insoluble materials were removed by ultracentrifugation (200,000g for 30 min at 4 C). The cell lysates were incubated with the PAcontaining or control liposomes at 4 C for 30 min. Samples were ultracentrifuged at 200,000g for 1 h at 4 C. The precipitate was dissolved in HEPES buffer. SDS-PAGE (15% acrylamide) was conducted, and separated proteins were detected by Western blotting using an anti-HSP27 antibody.
Confocal laser scanning microscopy COS-7 cells seeded on coverslips were transiently transfected with plasmids using PolyFect reagent (Qiagen) as described by the manufacturer. After 20 h of transfection, the cells were incubated with the DGKα selective inhibitor CU-3 (29) (or DMSO alone as a control) in DMEM (final concentration: 10 μM) for 30 min to inhibit PA generation by DGKα-CA. The cells were then fixed in 4% paraformaldehyde. The coverslips were mounted with Vectashield (Vector Laboratories, Burlingame, CA, USA). Fluorescence images were obtained with an Olympus FV1000-D (IX81) confocal laser scanning microscope (Olympus, Tokyo, Japan) equipped with a UPLSAPO 60 × 1.35 NA oil at room temperature. EGFP fluorescence was excited at 488 nm, and mCherry fluorescence was excited at 543 nm. Images were obtained using FV-10 ASW software (Olympus).

Blue native polyacrylamide gel electrophoresis
The purified 6× His-tagged HSP27 protein (final concentration: 2.3 μM) was dissolved in HEPES buffer and incubated with the PA-containing or control liposomes at 4 C for 30 min. BN-PAGE (4% stacking gel and 6% separation gel) was carried out as previously described (82). The gel was incubated in denature buffer A (20 mM Tris-HCl (pH 7.4), 150 mM glycine, and 0.1% SDS) for 10 min at room temperature. After transfer, polyvinylidene fluoride membranes were washed with methanol, followed by incubation in denature buffer B (50 mM Tris-HCl (pH 7.4), 2% SDS, 0.8% β-mercaptoethanol) for 30 min at 50 C. The proteins were detected using an anti-6× His antibody.

Statistical analysis
Data are represented as the means ± SD and were analyzed using one-way analysis of variance followed by Tukey's or Dunnett's post hoc test for multiple comparisons or two-tailed t test for the comparison of two groups using Prism 8 (GraphPad Software, San Diego, CA, USA) to determine any significant differences. p < 0.05 was considered significant.

Data availability
The data that support the findings of this study are available from the corresponding author [sakane@faculty.chiba-u.jp] upon reasonable request.